We aim to achieve nuclear reprogramming of somatic cells to endow them with pluripotent stem cell-like properties by delivery of the necessary transcription factors (Oct4, Sox2, c-Myc and Klf4) in cell-permeable form. These transcription factors will be equipped with cell-penetrating entities via the transpeptidation reaction carried out by bacterial sortases. This methodology should overcome the need of introducing the requisite transcription factors by transfection of DNA or by viral means of delivery, procedures that entail the risk of causing harmful genetic alterations in the recipient cells and so limit the therapeutic potential of pluripotent cells obtained by nuclear reprogramming. The bacterial sortases catalyze a transpeptidation reaction that allows the installation onto suitably tagged proteins of a wide variety of chemical modifications not accessible through genetic modification. The necessary chemical transformations are all readily accomplished by standard peptide synthesis and modifications thereof. The transpeptidation reaction recognizes the LPXTG sequence, which must be close to the C-terminus of the target protein, and results in the formation of an acyl enzyme intermediate between the sortase and the substrate, with concomitant release of the sequence C-terminal of the Thr residue that is part of the LPXTG motif. The acyl enzyme intermediate is resolved through nucleophilic attack of a peptide sequence that minimally consists of three Gly residues, onto which may be appended synthetically a large variety of different substituents. Alternatively, the target protein may be equipped at its N-terminus with an oligoglycine sequence that can serve as the nucleophile, so that it can attack a synthetic peptide that minimally consists of an LPXTG motif, to the N terminus of which one may attach, again, a large variety of different substituents through chemical synthesis. The design of these substituents will include a photocleavable entity or readily hydrolyzable ester linkage so that proteins, once delivered across the membrane to the cytoplasm, can be released from the cell- penetrating entities appended chemo-enzymatically. We propose to use Cre recombinase and electrophilic derivatives of ubiquitin as model protein substrates. Both of these proteins, if delivered successfully, impose an irreversible, readily detectable change on the cells that receive them and will serve as robust monitors of successful delivery. We then propose to install at the N-or C-terminus of the transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4 a cell-penetrating entity, to allow cytoplasmic and nuclear delivery of these transcription factors to somatic cells, for the purpose of achieving nuclear reprogramming and so impose an embryonic stem cell-like fate. The extent of reprogramming will be assessed by transcriptional profiling, and - ultimately- by generation of mice derived from these reprogrammed somatic cells. 7. PUBLIC HEALTH RELEVANCE The proposed research, if successful, would allow the generation of pluripotent stem cells with therapeutic potential. The proposed research will thus make an essential contribution to the development of cell based therapies for highly prevalent diseases, including muscular dystrophies, insulin-dependent diabetes, and neurodegenerative disorders.